Abstract
The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.
Highlights
From the Departments of $Molecular Genetics, Wmmunopharmacology, **Peptide Research, and 11Protein Biochemistry, Roche Research Center, Hoffman-LaRoche, Inc., Nutley, New Jersey 071I0
Sequence homologies, the 50-kDa a-subunitis a mem- Complementary DNA clones coding for the rat a (9, lo), p ber of the immunoglobulin superfamily of proteins and ( l l ), a n d y [12] subunits, the mouse a, p, and y-subunits has two predicted disulfide-bonded loops
Noninhibitory anti- The three a-subunits are members of the immunoglobulin bodies (i.e. 2237) immunoprecipitate bothlZ5I-labeled superfamily [9, 10, 13, 14], and the sequence for the human ch-a-protein and thseoluble complexof ‘251-IgEcross- a-subunit predictstwo disulfide-bonded loopswhich are comlinked to ch-a-protein bduot not block lZ5I-IgE bindinpgosed of amino acids51-93 and 132-176 of the full length ato thech-a-proteinexpressedon
Summary
Characterization of Monoclonal Anti-a-proteinAntibodiestibodies-Balblc mice (Charles River Laboratories) were immunized Mice injected with purified, active soluble ch-a-protein proby the intraperitoneal route with partiaplluyrified ch-a-protein 2 weeks later, the mice were injected intraperito- labeled ch-a-proteinand "'I-IgE. 7 weeks later, one mouse was injected intraperitoneallyandintravenouslywithch-a-protein(from 5.5 X shown). A fusion with spleen cells isolated from one mouse yielded 46 hybridomas secreting anti-a-protein antibodies. NSO cells [27] at a ratio of 1:l (spleen cel1s:NSO cells) with 35% (v/ preparation (Fig. 1).The two major radiolabeled proteins of v) polyethylene glycol 4000 -47 kDa and -65 kDa present in the lZ5I-ch-a-protein preparation represent labeled ch-a-protein and bovine serum albumin, respectively (Fig. l).Allof the prototype antibodies listed in Table I reacted with denatured and reduced ch-a-. Manuscript in preparation. protein on Western blots (data not shown)
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